Polymer Degradation and Stability 98 (2013) 542e549

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Polymer Degradation and Stability

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The effect of pH on the thermal stability of fibrous hard alpha-keratins

Dan Istrate a, Crisan Popescu a, b, *, Meriem Er Rafik a, Martin Möller a
a
DWI an der RWTH Aachen e.V., Forckenbeckstrs. 50, D-52056 Aachen, Germany
b
University “Aurel Vlaicu”, Bd. Revolutiei 77, RO-310130 Arad, Romania

a r t i c l e i n f o a b s t r a c t

Article history: The study of the effect of pH on the behaviour of proteins is still a topic of interest. We here report an
Received 15 March 2010 unexpected increase of thermal stability of hard alpha-keratins under the influence of pH that is
Received in revised form observed when investigating their thermal behaviour by differential scanning calorimetry (DSC). The
15 September 2012
results, particularly for oxidatively damaged keratin fibres treated with low pH solution, show a signif-
Accepted 4 December 2012
Available online 12 December 2012
icant increase of enthalpy and a shift of the peak temperature towards higher temperature for the
endothermic process assigned to the thermal denaturation. We propose a three-phase model for
describing the behaviour of fibrous hard alpha-keratins, in which the interface, made of non-helical tail
Keywords:
Keratin
domains of keratin, scaffolds the intermediate filaments and plays an important role. It is strengthen by
Thermal stability the action of strong acids and controls the thermal properties of the alpha-helix. The approach is sup-
Three-phase model ported by amino-acid analysis, X-ray diffraction, Raman spectroscopy and tensile strength observations.
Interface Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction governs the segmental mobility of the alpha-helix and as a conse-

Proteins exhibit their active properties within certain ranges of
values of temperature and pH and unfold beyond those limits.
While the optimal range of pH values may vary for various proteins,
the temperature interval is around 60e80
C for most of them [1].
A few proteins withstand temperatures above 100
C and among
them there are some of the fibrous proteins.
Fibrous proteins are distinguished from globular proteins by
their filamentous, elongated form. Most of them play structural
roles in animal cells and tissues. Among the most well known
representatives of this class are the alpha-keratins in human hair,
wool and finger nails, fibroin in silk, actin and myosin in muscles,
and collagen, the most abundant protein in vertebrate bodies [2].
Keratin fibres are described as being formed of crystalline rod-like
components made of alpha-helices (the intermediate filaments, IFs)
embedded in an amorphous matrix (keratin associated proteins,
KAPs) that have a relatively high amount of cystine ([3], and refer-
ences therein). This is known as the two-phase model and it is used to
explain the thermal and mechanical behaviour of keratin fibres [4,5].
The alpha-helix of soluble proteins denatures at temperatures
below 80
C. The fact that in insoluble keratins it denatures above
100
C is assumed to be due to the encapsulation of the helix in
a rigid matrix. The viscosity (crosslinking degree) of the matrix
* Corresponding author. DWI an der RWTH Aachen e.V., Forckenbeckstrs.
50, D-52056 Aachen, Germany. Tel.: þ49 241 8023300; fax: þ49 241 8023301.
E-mail address: popescu@dwi.rwth-aachen.de (C. Popescu).
quence the unfolding reaction (denaturation).
Little systematic work has been done on the thermal denatur-
ation of fibrous proteins, particularly keratins, because of their
being poorly soluble and difficult to purify in sufficient quantities
for biophysical studies. Over time the thermal denaturation of
keratin fibres was studied by DSC either in “dry”, or in “wet”
environment, respectively. The first method comprises investiga-
tions performed while allowing the moisture content of the keratin
sample to evaporate with increasing temperature. The “dry”
investigation shows the endothermic effect above 200
C, some-
times as a doublet [6e9]. Later evidence showed that the dry
experiments are very much obstructed by the pyrolysis reactions
occurring at similar temperatures and, therefore, ascribing the
endothermic peak to denaturation is not acceptable [10].
By the second method keratins are investigated in water excess,
in sealed pressure resistant capsules that keep the water during
heating [7,11e13]. This method shows the endothermic effect at
around 150
C. The enthalpy, DH, is assumed to reflect the amount
and structural integrity of the alpha-helical material in the IFs, and
the peak temperature, Tp, reflects the cross-link density of the
matrix in which the IFs are embedded [14].
Recently we showed that the breaking of disulphide bonds
between the IFs and the surrounding matrix is the limiting step of
the denaturation [15] and that a three-phase model better explains
the facts. 1H NMR results published previously further question
the validity of the two-phase model and support a three-phase
model [16].

0141-3910/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymdegradstab.2012.12.001
 D. Istrate et al. / Polymer Degradation and Stability 98 (2013) 542e549
The value of pH plays a major role for the activity of proteins and
significantly influences the denaturation of soluble proteins [17e19].
We expect that pH value also influences the denaturation of alpha-
keratins and the purpose of our work is to investigate this effect.
Understanding properly how the intermediate filaments of
keratins are protected against thermal denaturation until high
values of temperature, and the role played by the pH of the envi-
ronment in this process is of a clear interest for the fundamental
knowledge of protein denaturation, as it may suggest ways for
designing new high-temperature stable biomaterials.
2. Experimental methods
The alpha-keratin fibres used for analysis were of Caucasian
dark-brown hair, supplied by KERLING International Haarfabrik
GmbH. The fibres were cleaned with 1% Lauryl ether sulphate (LES)
and dried at room temperature prior to work with them. The pH of
their aqueous extract was found to be 6.5 to 7.
2.1. DSC measurements
The hair samples were cut into fine snippets (w2 mm) and
stored under controlled conditions (w24 h, 22
C, 55% relative
humidity) to ensure invariant water contents. 7e10 mg of each
sample were weighed and placed in crucibles.
Prior to sealing a crucible, 50 mL of distilled water (pH 6.7) was
added, and the sealed crucible was stored overnight (w14 h
preceding the measurement), to allow the fibres to wet.
The DSC experiments were run in a Perkin Elmer DSC-7, using
pressure resistant stainless steel large volume capsules. DSC cali-
bration was done with indium and palmitic acid, both of high
purity. The temperature ranged from 50 to 180
C at a heating rate
of 10 K/min. For each sample we performed 3e5 measurements
and the peak temperature, Tp, and enthalpy, DH, of the endothermic
effect are reported as mean values with standard deviations.
2.2. Tensile measurements
The tensile measurements were performed using a Miniature
Tensile Tester Model 675 (MTT675) and a Fibre Dimensional
Analysis Unit Model 765 (FDAS765) of Dia-Stron, UK. A minimum of
35 single fibres were tested for each sample at a stretching rate of
20 mm/min and a gauge force of 1 gf, as initial condition.
The measurements were performed in wet conditions, consid-
ered to reflect best the changes at the level of intermediate fila-
ments [20e22]. Prior to loading in the circular cassette, the samples
were immersed in distilled water for 120 min to allow them
wetting. During the measurements the cassettes were also filled
with distilled water to ensure the 100% humidity content during
the measurement.
The stressestrain curve recorded for each fibre allows calcu-
lating the Young’s modulus, the yield strength, the strain at break
and the total work, which characterise numerically the fibre
mechanics.
2.3. Amino acids analyses
Amino acids analyses were conducted conventionally on an
“Alpha Plus” Amino acid Analyser, manufactured by Pharmacia LKB,
Freiburg, Germany. The results are expressed in molar percentage.
2.4. X-ray microdiffraction
X-Ray microdiffraction experiments were performed at the
European Synchrotron Radiation Facility (Grenoble, France) on
543
microfocus Beamline ID13 [23]. A high intensity monochromatic
beam (wavelength k ¼ 0.961  A), coming from an undulator and a Si-
111 double crystal monochromator, was focussed with an ellip-
soidal mirror (focal spot 20 (h) * 40 (v) mm2) and then size-limited
down to a 5 mm diameter circular section by a collimator placed in
the focal plane. A guard aperture (PteIr, 10 mm diameter) reduced
diffuse scattering from the collimator exit. Samples were made of
10 hairs mounted on a frame with the hair axis perpendicular to the
X-ray beam on a computer-controlled gantry coupled with
a microscope which permitted sample positioning with a 0.1 mm
resolution.
The experiments were carried out with a 320 mm samplee
detector distance, which was calibrated using silver behenate, the
first order spacing of which is 58.38  A. Using a small beam stop
of 300 mm diameter, two-dimensional X-ray scattering patterns
were collected from 0.006 to 0.4  A. Patterns were recorded with
1 s exposure times on an MAR-CCD camera (16 bit readout;
130 mm entrance window; 2048  2048 pixels; pixel size of
78.94  78.94 mm2). Radiation damage of the structure has been
verified to only occur after exposure times longer than 10 s and it is
indicated by the strong weakening and broadening of the scattering
features. About five patterns were collected along each hair.
Data analysis focussed on the scattering regions that provide
information for the various structural organization levels, viz.:
(i) the meridian arc located in the 5 A region, which is produced
by the regular alpha-helical coiled-coil packing. The strong
intensity of this arc was shown to relate to the fine configu-
ration of residues [24e26].
(ii) the fine meridian scattering arc at 67  A; this is indicative for
the periodic architecture of the molecules along the IF.
(iii) the equatorial small-angle X-ray scattering zone, which is
related to the radial geometry of the filaments and to their
lateral packing organization in the matrix. In particular, the
distorted crystalline lateral organization gives rise to a strong
equatorial reflection observed around 90  A [27e29].
The analysis was carried out following two complementary
procedures. The position and intensity of the main scattering
features were first estimated and compared by a visual inspection
of all patterns. For the most representative patterns, one-
dimensional equatorial profiles passing through the origin, both
along the equator and the meridian, were extracted from the
two-dimensional patterns by integrating the intensity around the
equator on a 10 pixels thick rectangular strip. These profiles yield
the precise positions and intensities of the main scattering
features and make the comparison of patterns and modelled
profiles straightforward. In the SAXS zone the huge scattering
intensity (proportional to S2.3) comes from nonkeratinous zones
in hair [27]. This component has been subtracted from the
profile.
2.5. Raman spectra
Raman spectra were obtained using a Bruker RFS-100 Raman
spectrometer equipped with an Adlas Nd:YAG laser of 1064 nm
wavelength operating at 200 mW. A bundle of keratin fibres was
fixed on an aluminium support, the Raman spectra being recorded
for the entire bundle.
Data acquisition and analysis was performed using Bruker OPUS
software (version 4.0). Raman spectra were collected at a resolution
of 4 cm1 and a modulation frequency of 5.0 kHz. We ensure that
no sample degradation occurred and that the obtained spectrum is
reproducible by collecting three spectra from each sample, and by
taking their average.
544 D. Istrate et al. / Polymer Degradation and Stability 98 (2013) 542e549

The bands of particular interest lie within the wave number 115
155.9 C
range of 500e1800 cm1. These are vibrations assigned to the SeS Δ H = 14.2 J/g
110
and CeS bonds of cystine, the amino acids (tryptophan, tyrosine, pH = 2
and phenylalanine), the amide I, and amide III vibrations and the 105
CeC skeletal stretching vibration of the a-helix.
The disulphide (SeS) content of the keratin samples was 100

Heat Flow / mW
150.5 C
compared by estimating the ratio of the peak area of the SeS band
95
(calculated from the peak to a baseline which was drawn between pH = 7 Δ H = 14.7 J/g

470 and 560 cm1) divided by the peak area of the CeH band 90
(calculated from the peak to a baseline which was drawn between
1375 and 1500 cm1) [30]. Also, the component content at 85

1671 cm1 assigned to the beta-sheet and/or random coil forms and 80
the component content at 1652 cm1 assigned to the alpha-helix 143.7 C
form of the hair samples was compared by estimating the ratio 75
pH = 11 Δ H = 11.7 J/g
of the peak area of each component divided by the peak area of the
70
CeH band (calculated from the peak to a baseline that was drawn 130 135 140 145 150 155 160 165 170
between 1375 and 1500 cm1) as described by Kuzuhara [30]. Temperature / C

2.6. Treatments Fig. 1. Endothermic peaks recorded on DSC for untreated hair at pH 7, and hair treated
with pH 2 and pH 11.

Damaging: the damage of the keratin fibres was induced by an

oxidative treatment with commercial bleaching products (IGORA
VARIO BLOND PLUS bleaching powder and IGORA ROYAL 20 vol 6%
H2O2 bleaching lotion) kindly supplied by Schwarzkopf. The
oxidative procedure followed the instructions of use. The fibres
were thoroughly rinsed after treatment and the pH of the aqueous
extract was checked to be 7.
pH treatment: the hair tresses were immersed in aqueous
solutions at a liquor ratio of 1 g fibres to 200 mL solution with
different values of pH, for 30 min at room temperature. The fibres
were then rinsed under tap water for 3 min, washed with a solution
of the IFs were damaged as a result of high pH value and there is
less crystalline material to denature thermally.
The effect of low pH is difficult to understand. According to the
two-phase model of keratins the increase of peak temperature and
of the enthalpy, as observed with strong acid treatment, means the
a
165
160

of Texapon N70, 0.1 mL/L, (70% sodium lauryl sulfate), rinsed with 155
Peak temperature, Tp / ° C

warm water for 1 min, then with tap water for 3 min, and immersed
overnight in distilled water to completely neutralise. The fibres 150
Untreated reference sample
were finally dried under a hot air flow. 145
We used acetic acid for adjusting pH from 1 to 7, ammonia for
the values from 7 to 12 and NaOH for pH 13. 140

135
3. Results and discussions
Oxidatively damaged reference sample
130

The DSC curves recorded for a sample of untreated hair (pH 7), 125
and treated with pH 2, and pH 11, respectively, are shown in Fig. 1, 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
as an example. pH value of treatment
The plot of untreated hair exhibits the characteristic endo- b
thermic effect around 150
C, with an enthalpy (area under the
20
peak) of about 15 J/g. As it is noticeable from Fig. 1 the endothermic
effect shifts with the nature of the treatment towards lower or
higher temperatures, and the area below peak changes too.
The pH is known to have a major influence on the denaturation
Enthalpy, DH (J/g)

15
of soluble proteins [16e18] and Fig. 1 indicates that it also plays
Untreated reference sample
a major role during thermal denaturation of hard alpha-keratins.
Fig. 2a and b summarises the measured influence of a relatively
short pH treatment on the recorded wet-DSC parameters of
10
Caucasian hair samples. One notices that the temperature of the
peak, Tp, shifts with approximately 5
C towards higher values, and Oxidatively damaged reference sample
that the value of enthalpy increases also as a result of treatments
with low pH solutions on untreated fibres (diamond symbols); the
high value of pH (alkaline treatment) has an opposite effect. 5
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Alkaline pH is favourable to cystine disruption and to the pH value of treatment
hydrolysis of the amide bonds and formation of lanthionine and
lysinoalanine [31,32]. This may explain the variation of both DH and Fig. 2. Peak temperature, Tp, (a) and value of enthalpy, DH, (b) with standard devia-
Tp as a result of alkaline pH treatment. Breaking of the cystine tions for wet DSC experiments recorded at 10 K/min after a pH treatment of keratin
fibre samples. The diamonds refer to the non-damaged material and squares to the
disulphide bond, located preponderantly in the matrix, leads to the oxidatively damaged keratin fibres. The references, marked also with bands across the
decrease of viscosity and the decrease of Tp value as a consequence; Figure, both for non-damaged and for the oxidatively damaged keratin have the pH of
the decrease of enthalpy indicates that part of the helical segments the aqueous extract of 7.
 D. Istrate et al. / Polymer Degradation and Stability 98 (2013) 542e549 545

reorganization and the folding of parts of originally amorphous Table 2

matrix. Amino acids composition (mol%) of bleached 3 times keratin fibre sample at pH 7
and bleached 3 times keratin fibre samples subsequently submitted to a pH3
Even more striking results were obtained after a relatively short treatment.
time exposure to low pH values of a keratin material previously
damaged by an oxidative treatment (see the squares in Fig. 2a and Amino acid 3 3 & pH 3 Amino acid 3 3 & pH 3

b). A strong increase of almost 30
C of the peak temperature of Cysteic acid 4.88 4.88 Methionine 0.19 0.21
Aspartic acid 5.88 5.98 Isoleucine 2.66 2.74
oxidative damaged samples subjected further to an acid treatment
Threonine 8.24 8.51 Leucine 6.36 6.40
at pH ranging from 1 to 3 is readily noticeable. This is at odds with Serine 12.07 12.15 Tyrosine 1.38 1.32
the two-phase model according to which a decrease of both peak Glutamic acid 12.88 12.68 Phenylalanine 1.84 1.83
temperature and enthalpy indicates an irreversible damage of the Proline 7.91 7.71 Ornithine 0.09 0.12
alpha-helix. As data of Fig. 2 suggest, the decrease of both values, Glycine 6.26 6.24 Lysine 2.88 2.83
Alanine 4.67 4.83 Lanthionine 0.00 e
recorded after the oxidative treatment, seems to be well reversed Valine 6.12 6.20 Histidine 1.02 1.05
by pH treatment. Cystine 7.68 7.33 Arginine 6.97 7.05
The duration of treatment with pH solution does not play
a significant role for increasing the thermal stability, as the results
of Table 1 indicate. It appears that adjusting the pH of the water in
the pan of DSC experiment prior to heating leads to similar results structure of hard alpha-keratin in hair shaft is inferred from X-ray
with a 15e30 min separate treatment. scattering and electron microscopy analyses. Along the meridian
As mentioned, within the framework of the two-phase model axis (fibre axis), the dimmers characterized by a regular alpha-
the unexpected increase of the thermal stability of samples treated helical coiled-coil folding in the rod central domain give rise to
with very acidic pH is understood to be due to the reorganization the wide-angle X-ray scattering (WAXS) meridian arc located in the
and the folding of parts of originally amorphous matrix. We have 5.15 
A region. The strong intensity of this arc was shown to relate to
investigated, therefore, the changes of fibre structure as a result of the fine configuration of residues [25,26]. At the small-angle X-ray
the treatment. scattering (SAXS) region the strong and fine 67  A meridian scat-
Firstly we have analysed the amino-acid composition of the tering arc is related to an axial stagger of molecules or group of
samples. Table 2 shows that the amino-acid composition of the molecules along the intermediate filament, IF [34e36]; its position
sample before and after exposure to strong acid (low pH value) is almost insensitive to humidity variations [35,37]. Along the
does not differ significantly, indicating that the acidic treatment equatorial axis, the X-ray pattern gives poor information about the
does not hydrolyse the protein chain. intermediate scale arrangement of the chains inside IFs. For the
We also do not notice any difference (within experimental WAXS equatorial region, the broad scattering maximum located at
error) of the total amount of SeS content of the fibre as a conse- 9.5 A peak is supposed to be due to interferences of coiled coil
quence of exposure to acid. chains [28,29] or to the distance of chains from others structures
It is accepted that the tensile properties of the fibre in the wet [38]. In the SAXS equatorial region the three broad peaks corre-
state are largely controlled by the properties of the intermediate sponding to the distances of 90  A, of 45 
A, and of 28 A, respectively,
filaments, because IFs are water-impenetrable, while the matrix is (located at S ¼ 0.012, 0.022, and 0.036 Å1) are provided from the
softened by water [5,20,22]. As a consequence the variation of the dense lateral IF packing. Hair contains crystallized lipids [39], more
wet tensile strength should correlate with the change of enthalpy, precisely soaps [40], that give rise to a series of rings, of which the
and the increase of the enthalpy value should reflect in an increase first order is superimposed on the peak at 45  A. The signals due to
of the tensile strength. soaps are the only variable scattering signals displayed by hair; the
Fig. 3 summarises the influence of the pH treatment on the wet signals due to keratin are fairly sample-independent. The pio-
tensile strength. The overall aspect of the stressestrain curves does neering X-ray scattering analyses of Fraser have established that
not change with the treatment and it is similar to the results re- the IFs are located at the nodes of a distorted two-dimensional
ported in literature [3,5]. In spite of the large change recorded on quasi-crystalline array [28,29]. This model was later refined using
DSC plot after a short pH exposure of hair, the fibre tensile strength an analytical description of the corresponding small-angle X-ray
(Young’s modulus) is not affected. This is supported by the amino- scattering (SAXS) equatorial X-ray scattering pattern [28]. So, the
acid analysis which did not show any break of covalent bonds or dense lateral packing of the intermediate filaments embedded in
formation of new cross-links of the keratin. The value of the yield the matrix namely the IF-matrix network can be investigated in this
strength (the stress at which the fibre material begins to deform region. The position and the intensities of these peaks are charac-
plastically), varies also insignificantly. Summing up, the treatment teristics of IFs diameter and of the mean of the centre-to-centre
with low pH solution does not change significantly the tensile distance between IFs [35]; when the hair fibre is immersed in
strength of the keratin material. water, the 90  A peak position increases indicating the matrix
The X-ray diffraction patterns of hard alpha-keratins are of swelling [35,41].
central importance in structural studies of this material [33]. The Fig. 4a shows that with the exception of an increased disorder
degree between the IFs, in the WAXS region (from 0.22 to
Table 1
0.066 Å1) the coiled coil configurations remain virtually unaf-
Variations of DSC parameters recorded for keratin fibres bleached 3 times and fected by acid treatment of keratin structure. As it appears, there is
immersed in water of pH 3 in DSC pan. The capsules were kept for t1, t2, and t3 (min) no change of the crystalline degree to account for the recorded
respectively before starting the heating. The values of untreated hair were recorded increase of enthalpy. The high intensity peak at 66  A of the sample
at pH 7. Data recorded with heating rate of 10 K/min.
of three times bleached hair and kept at pH 1 is probably a parasitic
Sample Tp  st. dev. (
C) DH  st. dev. (J/g) scattering effect of the air. This point of view is also supported by
Untreated 150.4  0.3 14.7  0.2 the absence of large intensity differences at the peak from 5.17  A.
Bleached 128.6  0.7 9.8  0.8 In the SAXS zone (from 0.066 to 0.01 Å1) given in Fig. 4b, the
t1 ¼ 15 159.8  1.0 13.1  1.4 huge scattering intensity (proportional to S2.3) was shown to
t2 ¼ 5 158.7  0.8 12.6  0.6
t3 ¼ 0 159.4  0.7 12.7  0.5
proceed from nonkeratinous zones in hair [27]. This component has
been subtracted from profile. The reflection at 45  A is provided by
546 D. Istrate et al. / Polymer Degradation and Stability 98 (2013) 542e549
Fig. 3. Wet tensile properties recorded after pH treatment of non-damaged keratin fibre, respectively bleached, as “box & whisker” plots, characterized by arithmetic means, the
standard errors (box) and the expectation ranges for the 95% confidence limits (whisker). (N) refers to untreated material and (B) to previously bleached damaged keratin, both at
pH 7.
crystallised lipids. The calculation of inter-IFs distances gives
93.121  A for native hair and 90.211  A for hair kept at pH 1 (both
native and bleached 3 times) respectively, while the diameter of the
IF was found of 37.5  A in all cases. Furthermore, from the lateral
organisation (i.e., equatorial intensities), no significant changes
were recorded for the 90  A peak position. Consequently we may
conclude that there is no significant change of the fibre structure
(matrix swelling or shrinking, increase or decrease of the amount of
IFs) to justify the shift of the peak temperature, Tp, or enthalpy, DH,
with the recorded magnitudes.
Raman spectroscopy provides information about the status of
SeS groups and about the state of ordered, respectively disordered
proteins.
White keratin fibres which followed the same damaging by
oxidative treatment (bleaching) and pH treatment as the brown
fibres were used as sentinel sample in order to prevent the fluo-
rescence from melanin.
The Raman spectra of bleached 3 times and bleached 3 times
and pH 1 treated samples are shown in Fig. 5.
One may notice that the intensity of the band assigned to SeS
bond does not change significantly, indicating that the disulphide
groups do not reform following the acid treatment. The SeO band
intensity at 1040 cm1, assigned to cysteic acid [30], remained also
unchanged.
No shift of the peak or modification of the area were noticed as
a result of low pH treatment for the band at 1671 cm1 and
assigned to the beta-sheet and/or random coil form, and for the
band component at 1652 cm1 assigned to the alpha-helix form
[30]. Also the amide III (unordered) band intensity at 1243 cm1,
assigned to the random coil form does not change significantly to
explain the shift of the DSC recorded peak temperature, Tp, or
enthalpy, DH in terms of formation of new crystalline structure.
All these point out that the increase of thermal stability of
keratins observed after a low pH treatment is likely to be due to
a change of the environment of the intermediate filaments, rather
than a change of the amount of crystalline material in the IFs.
Since a model with two separated phases cannot explain these
data we consider a variant in which the nonhelical terminal
domains that project into the interfilamentous space and link with
the matrix proteins control and enhance the thermal properties of
keratin filaments. In other words we propose that the interface
phase, lying between crystalline and amorphous phase, plays
a more important role than it was assumed so far. This is in line
with our previously reported results on the kinetic mechanism of
thermal denaturation of keratins which considers the degrading of
an interface phase as the rate-determining step of thermal dena-
turation of the alpha-helix (IFs) [15]. As it was shown, a keratin fibre
for which the interface is completely damaged records under same
heating rate of 10 K/min in DSC the peak temperature, Tp, at around
82e84
C and the enthalpy of about 8 J/g [15]. Moreover, results
reported on NMR investigation of the keratin fibres indicated the
strong adsorption of protons (water molecules) at the interface of
the intermediate filaments (IFs), supporting also the shielding role
of the interface as we infer from these experiments [16].
Fig. 6 gives a simplified schema of a microfibril with protofibrils
showing the alpha-helical rods and the nonhelical terminal
domains projecting into the interfilamentous space and linking
with the matrix proteins through disulphide bonds, as suggested by
Chapman and Hearle’s model [43]. The terminal domains contain,
besides cystine, glycine, threonine, valine, alanine and serine, acidic
sites as glutamic and aspartic acid [32]. This way the electrostatic
forces may well play a role for the stability of IFs in native fibre. In
our view, this scaffolding structure at the IFs surface made by the
side-chain interactions that anchor microfibrils to matrix, and
named ‘interface phase’, assists the thermal stability and the
primary control over the denaturation of helical material of kera-
tins when heated. We consider, in line with our previous results
[15,16] and with what Crewther and Hearle suggested [42,43], that
 D. Istrate et al. / Polymer Degradation and Stability 98 (2013) 542e549
Fig. 4. Changes in meridian (a) and equatorial (b) intensities of X-ray pattern of
untreated and three times bleached keratin fibres samples after 30 min exposure to pH 1.
the matrix structure is made of mainly globular proteins with
a high density of disulphide cross-links. It has a protective role and
the capacity to participate to the formation of a strong interface
protecting the intermediate filaments.
The model allows explaining the effect of pH on thermal
denaturation of hard alpha-keratins in terms of mechanism of ions
(ligand) binding. The ligands are the aqueous protons (Hþ) that
bind to the specific sites in both folded and unfolded states of the
Fig. 5. Raman spectra of bleached three times, and bleached three times and pH 1
treated white keratin fibres.
547
Fig. 6. A 3-D structure of hard-alpha keratin intermediate filament embedded in the
matrix, build for giving account to thermal behaviour of the keratinous fibres. The
central rod domain is dominated by a-helical subsegments (1A, 1B, 2A, 2B, graphed as
small rectangles inside the rod) separated by short linker regions (L1, L12, L2, graphed
as lines between the rectangles). The rod is flanked by nonhelical head and tail
domains at the NH2e and COOHe termini that extend into the matrix through cystine
bonds and link with the matrix proteins. Together with other linkages, the interface, as
the scaffolding structure at the surface of IFs, controls and enhances the thermal
properties of keratin filaments. For the sake of clarity, some terminal domains
projections are omitted.
IFs and KAPs, in a similar manner as in the case of soluble proteins
[44]. The plateau recorded for peak temperature, Tp, when pH
ranges from 1 to 3 (see Fig. 2a), implies that both IFs and KAPs
phases are fully liganded, the interface reaching the maximum
cross-link density attainable under the given conditions.
The heat of the endothermic process recorded by DSC for the
denaturing of fibrous keratins is a cumulative effect of all partici-
pating groups. The observed increase of DH arises from the addi-
tional energy required to remove the ligand prior to unfolding of
548 D. Istrate et al. / Polymer Degradation and Stability 98 (2013) 542e549
the helical components and thermal denaturation of the IFs.
Analogous, a decrease of enthalpy as a result of damaging treat-
ments, which does not reflect in a change of the X-ray diffraction
pattern, is likely due to interface weakening, rather than to
a change of fibre crystalline degree [15].
As it appears, the interface is playing the role of a shield, or
a scaffold for the intermediate filaments. The shield rigidity is tuned
with aqueous protons concentration and it controls the thermal
denaturation behaviour of the intermediate filaments after the
matrix fails.
The SeS cross-links from the interface also influence the
denaturation. As shown schematically in Fig. 6 the disulphide
bonds anchor the interface on the matrix and any damage of the
scaffold required for inducing the denaturation needs ultimately
the breaking of these bonds.
The mechanism of thermal denaturation of keratins, as we
showed elsewhere [15], follows several steps. Beyond a certain
temperature, the temperature rise lead to the break of disulphide
bonds which keep the IFs embedded in matrix. Once set free, the IFs
denature. This inevitably involves a transition from a relatively
compact ordered structure to a more flexible, disorganized, open
polypeptide chain. As the denaturation proceeds the protein
molecules unfold and the intern hydrophobic regions expose to the
outside of the molecules. The hydrophobic groups in water tend to
cluster, leading to associates of molecules. This is only possible after
the polypeptide chains are set free from the proposed scaffold
structure. Since the covalent SeS bonds have the highest energy
amongst the bonds which control the interface, their break is the
rate determining step of the denaturation. The total heat recorded
(DH) is the sum of all those heats required for the stability of the IF
structure, namely the heat for removing the ligands of the interface,
the heat for breaking existing di-sulphide bonds and the heat for
denaturing the alpha-helix.
Summing up, the proposed model describes the fibrous keratins
as comprised of an amorphous matrix, a crystalline phase and an
interface between the two phases. The role of the interface appears
more important for thermal behaviour than for the mechanics of
the fibre. The thermal denaturation of alpha-keratins requires, as
a rate determined step, the breaking of the scaffold structure, for
setting free the a-helical domains which unfold.
4. Conclusions
We report a strong influence of the pH of the treatment
(particularly low pH values) on keratin fibre thermal stability as
measured by DSC experiments in water. Following short applica-
tion of a low pH (below 3) solution the thermal stability of the
keratins may improve by at least 5
C. The amino acid analysis,
tensile measurements, X-ray analysis and Raman spectroscopy do
not indicate significant changes of chemistry or crystallinity of the
keratin to account for the shifts of peak temperature and value of
enthalpy of the endothermic process. Based on these results and in
line with our previous works [15,16] we propose a three-phase
model for keratin fibres, in which the nonhelical (or globular)
terminal domains of keratin promote filament interactions and
control the thermal properties of keratin intermediate filaments.
The interface phase, whose strength is increased at low values of
pH, scaffolds the intermediate filaments and controls this way the
thermal stability. The thermal denaturation of the intermediate
filaments can occur only after the scaffold is damaged irreversibly.
Acknowledgements
The financial support for this work from Beiersdorf AG, CIBA
Spezialitaetenchemie Grenzach GmbH, COGNIS GmbH, HENKEL AG
& Co. KGaA, KPSS-KAO Professional Salon Services GmbH and
WELLA Services GmbH through the German cosmetic industry
committee “DSC of Human Hair” is gratefully acknowledged.
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